Transactivation of receptor tyrosine kinase (RTK)-regulated signaling pathways following G protein-coupled receptor (GPCR) activation has recently been described and can occur through intracellular and extracellular mediators. Activation of RTK signaling pathways by GPCR has explained some of the cell proliferative effects associated with GPCR agonists. We have discovered a novel mechanism by which an RTK agonist potentiates GPCR-mediated signal transduction; specifically, pretreatment of rat intestinal epithelial cells with epidermal growth factor (EGF) increases bombesin (BBS)-stimulated Ca2+ release from intracellular Ca2+ stores in a dose-dependent manner. BBS stimulates Ca2+ release from the inositol 1,4,5-triphosphate (lnsP3)-sensitive Ca2+ stores mediated by the gastrin-releasing peptide receptor (GRP-R), a member of the GPCR superfamily. The stimulatory effect of EGF on GRP-R-mediated Ca2+ release requires activation of the mitogen-activated kinase kinase (MEK)-1,2/extracellular signal-regulated kinase (ERK)-1,2 pathway. Reduction of basal MEK-1,2/ERK-l ,2 activity by either serum starvation, treatment with selective MEK-1,2 inhibitors or expression of a dominant-negative mutant form of MEK-1 decrease BBS-stimulated Ca2+ release from intracellular stores. Conversely, increasing basal activity of the MEK-1,2/ERK-1,2 pathway reversed the inhibitory effects of serum starvation on G17-stimulated Ca2+ release. These data demonstrate a novel role for basal MAPK activity in the regulation of Gl peptide hormone-mediated Ca2+mobilization and suggest a potentially important mechanism for "cross-talk" between the GPCR-regulated pathways and regulators of MAPK activity, such as EGF. We hypothesize that MAPKs play a central role in signal integration, in part, by modulating the sensitivity of Gl peptide hormone receptor-mediated response to agonist stimulation and by coupling growth factor receptor kinases to Gl peptide hormone-regulated signaling pathways. Our Specific Aims are: 1) To determine the molecular mechanisms by which mitogen-activate protein kinases (MAPKs) modulate Gl peptide hormone-mediated increases in [Ca2+]j. 2) To determine the effects of modulating basal MAPK activity on GRP-R-regulated gene expression, cell proliferation and peptide secretion. Uncovering the molecular mechanisms involved in MAPK sensitization of Gl peptide hormone receptor-mediated signal transduction will get us closer to realizing the full therapeutic potential of Gl peptide hormones in human disease.